Kumar A.

et al: Pepsin Assay for Prescreening of HIV-Protease Inhibitors

Journal of Pharmaceutical and Scientific Innovation

www.jpsionline.com
Research Article
PEPSIN ASSAY ONE OF THE EASIEST APPROACH FOR PRESCREENING OF HIV-PROTEASE INHIBITORS
Singh K.P.1, Kumar A.2*, Prasad R.3
1
Junior research fellow, Dept. of Botany, University of Rajasthan, Jaipur
2
Professor, Dept. of Botany, University of Rajasthan, Jaipur
3
Associate professor, Dept. of Biotechnology, IIT Roorkee; India
*Email: msku4@hotmail.com
Received on: 04/12/12 Revised on: 25/02/13 Accepted on: 28/02/13
ABSTRACT
As HIV is quickly becoming one of the world deadliest viruses, one of the biggest problem in curing AIDS is as HIV can easily develop resistance against the
provided drugs. HAART (Highly active antiretroviral therapy) is the only one hope for the AIDS patients till now, which can increase the survival period of
the AIDS patient by sustaining the viral load below 50 copies/ml. in blood serum, in which we use combination of three to five drugs targeting different stages
of replication cycle of HIV. HIV-protease inhibitors are the indispensable part of this therapy. There is a great need of screening of anti-HIV agents from
chemical as well as natural resources. But this process is not so easy because it needs huge amount of money for required chemicals, as well as highly
sophisticated labs. Here we have developed a substitute of this problem unto some extent, i.e." Pepsin Assay", it is a substitute of HIV- protease, both of them
belongs to same Aspartyl family of enzyme & share same signature sequence at the active site. So we hope that development of this assay will enhance the
screening programme of HIV-protease inhibitors.
KEY WORDS: HIV-protease inhibitors, HAART, Pepstatine A, STI, AIDS, CD4+ Cells.

INTRODUCTION
A number of laboratories in the world are actively involved
for the investigation of anti-HIV agents that interfere with
different stages of HIV replication cycle. There are still no
cure & no vaccine for AIDS. But treatment with a
combination of two nucleoside analogues and a protease
inhibitor (triple or combination therapy) has been in use since
1994 and remarkably successful in halting virus replication
and the progression of AIDS. There are many evidence of
research in support of efficacy of combination therapy1-4.
This therapy is now known as highly active antiretroviral
therapy (HAART), however viral level in plasma reaches up
to an undetectable level under a successful triple therapy (i.e.
upto < 50 copies/ml.), still HAART unable to clear the virus
completely from the AIDS patients. There is a latent virus
present in the resting CD4+ memory cells and other
reservoirs as well, so the treatment must be continued without
a long break, long term break cause the virus to rebound
within a few days to its original level, but prolonged
exposure to a particular drug cause the development of the
resistance against the provided drug, some researchers
suggest that short term break in treatment almost always lead
to viral rebound that can be re-suppressed with HAART;
immune response to HIV improves. This therapy also called
Structured Treatment Interruption (STI)5. There are many
other problems with the conventional chemotherapy provided
for the treatment of AIDS, the clinical trials for the
administration of these compounds to the AIDS patients
revealed serious side effects. So there is a great need of
development or screen out different compounds having antiHIV activity with minimum toxic effects, which relates to
different pathways of HIV infection, should prove important
to prevent disease progression. It seems that a better strategy
to find novel antiviral agents with less cytotoxicity is to look
for natural substances. Many reviews on medicinal plants
show the importance of natural products for curing many
highly threatening diseases such as cancer, AIDS etc. Singh

I.P. et. Al. reviewed natural anti-HIV products targeting

different stages of HIV life cycle6.
Over the last decade, antiviral researchers have also turned
too many of the traditional folk medicines, invariably a
cocktail of natural products, to uncover the scientific basis
of their remedial effects. Recently review published plantderived anti-HIV7-9 compounds, which serves to underline the
fact that selected medicinal plants with HIV-inhibitory
activity are widely distributed in nature10-11.
HIV-protease is the indispensable part of HAART therapy, as
it plays a significant role in the HIV replication cycle. HIV
protease was first suggested as a potential target AIDS
therapy by Kramer et.al. 198612. After that it was shown a
frame shift mutation in the protease region of the pole-gene
prevented the cleavage of the Gag-Pole polyprotein
precursor, which is the essential process for the maturation of
structural & functional viral protein. Blockage of HIV
protease leads to the formation of immature non-infectious
virions. Compounds having ability to inhibit this protease
have been studied intensively during the last decade and
numerous reports have been published, Alterman, M
2001gave a comprehensive summery of design and synthesis
of HIV-1 Protease inhibitors13. Saquinavir was the first
approved protease inhibitor & has been in clinical use since
199514, presently there are six clinically approved protease
inhibitors in the market available, although the inhibitors in
the market are highly selective they induce side effects such
as lipodystrophy, hyperlipidaemia, insulin resistance and
emergence of the resistant mutants upon prolonged use of a
particular drug therefore there is probably be a constant
demand of new HIV protease inhibitors. Now the medical
science community turned towards the extracting natural
resources like plants15-17, herbs18, marine algae19 and many
other organisms for those bioactive compounds by which we
can cure so many human disorders and pathogenic infections
including AIDS and cancer. Here we developed a indirect
approach for screening of HIV protease inhibitors from
natural products.
JPSI 2 (1), Jan Feb 2013, 53-56

Kumar A. et al: Pepsin Assay for Prescreening of HIV-Protease Inhibitors

HIV-Protease
Enzyme such as HIV-protease, natures own catalysts,
proteases are a divers class of enzymes that catalyze the
cleavage of peptide or proteins. Based on the presence of the
characteristic signature amino acid sequence Asp-Thr-Gly. It
was suggested by Toh et al in 1985 that protease of HIV
might be belongs to the family of Aspartic proteases20. This
was confirmed through Pepstatine A inhibition, an aspartic
protease selective inhibitor21 and by sit directed mutagenesis
of the active site Asp-25, which led to abolition of the
catalytic activity22-23. the aspartic proteases are the well
characterized group of enzyme that can be found in
vertebrates, plant, in addition to in fungi, example of
proteases from the Aspartic protease class are- Pepsin,
Cathepsin D, Renin, Chymosin, Penicillopepsin and
Rhizopus pepsin; which all are two domain enzyme with
more than 300 residues in length and contain the Asp-ThrGly sequence in each domain that form the active site, which
effectuate the cleavage reaction since the HIV protease
sequence is no more than 99 amino acids and contains only
one one the required triad Asp-Thr-Gly it was suggested that
the active form of the HIV protease was a homodimer of 198
amino acids24. This hypothesis was later confirmed by X-ray
crystallographic determination25-26. As both HIV-protease &
pepsin share the signature sequence Asp-Thr-Gly, an overall
similarity of primary structure, inhibition by pepstatine A and
are inactivated by mutation of the putative active -site
aspartates therefore these all data suggests that pepsin may be
a representative for the HIV protease in the aspartic group27.
So pepsin assay could be used for pre-screening of HIV
protease inhibitors. Maria del et.al 2004 also used the assay
system to screen out HIV-protease inhibitor from Rapseed
protein hydrolysate28. A detailed Computational Studies on
HIV-1 Protease Inhibitors was performed by Wesley
Schaal29.
MATERIAL & METHODS
Chemical required
Enzyme Pepsin (Himedia), Hemoglobin (Himedia), plants
extract, Sodium Acetate tri hydrates (Qualigens), Sodium
Chloride (Himedia), Acetic acid, Tri Chloro Acetic acid
(TCA) (Sdfine) and experimental plant material.
Instruments required
Spectrophotometer - Karry 100, Sigma table centrifuge for
14000 rpm, Rami for 8000 rpm, Micropipette 5-40 l, 40200 l, 200-1000 l, Eppendorff (1.75ml), Mixer grinder,
Mortar pestle, electronic balance.
Plants extract preparation
Many plants were selected for our study, out of which
Aspargus recemosus, Zingiber officinele, Aloe barbadensis
plants give more significant results. Different parts of fresh
plant material were taken, washed properly under tap water, 5
gm of each were taken, Cut it into small pieces, Ground it
properly in mortar and pestle, 10 ml of DW were added to
get fine paste, Centrifuge for 30 min. at 8000 rpm.
Supernatant were collected & pallet were discarded and
supernatant was used as a crud extract of plant material.
Enzyme Pepsin activity inhibition Assay: - pepsin have a
quite close resemblance in proteolytic activity with HIV-1
protease one of key enzyme of HIV-1 life cycle as both of
them belongs to same Aspartate enzyme family 28. So here
we use this enzyme as a substitute of HIV-1 protease to check
out anti HIV activity of plants extract.

Procedure
We have done slight modification in this assay system as
mentioned by Maria del et.al. in pepsin assay the ref paper,
we have taken almost double quantity of all constituents as
well as reaction mixture and here we use crud plant extract in
place of known amount of partially purified plant extract as
used in previous works.
For that assay we have taken 50g pepsin, 800g
hemoglobin and crud plant extract in 500l of reaction
mixture. Allowed to incubate for 20 min. at 370C, 700l of
5% TCA was added to stop the reaction, after few min.,
centrifuge at 14000 g for 5 min. and supernatant was
collected and O.D. was taken at 280 nm.
Separate blank were used for both positive and negative
control as well as for sample. For positive control we have
taken enzyme and substrate and follow the above procedure,
and for negative control we have taken pepstatin as a well
known inhibitor of both Pepsin and HIV-Protease. And for
testing sample we have taken extract in place of pepstain. We
have taken four plants for that study which gives almost same
results with several repetitions, so this assay gives
reproducible results.
Several experiments were performed to get optimum activity
of enzyme:
Parameter - optimum range
pH - 2- 4
Incubation temperature - 37 0 C
Incubation period - 20min.
Centrifugation - 14000 g.
Reaction volume - 500 L
Scanning of Hemoglobin solution was performed in the range
of 150 to 650 nm. Gives three peaks, first at 206 nm, second
at 280 and third at 410 nm (figure-1). Highest peak at 206
nm denote most probably the presence of large number of
peptide bonds in Hemoglobin protein, peak at 280 must be
due to the presence of tyrosin and tryptophan amino acids
and peak at 410 nm might be due to the presence of Heam
group in that protein. But here we choose peak at 280 nm for
our experimental observations.

Fig. 1: scanning of Hemoglobin shows three peaks. At 206 at, at 280 and at
410 nm

We performed another experiment for pepsin enzyme

kinetics; here we got a straight line (Fig.-2) while increasing
concentration of hemoglobin within the range of 200- 800 g
above that a drop in slop had been observed and keeping
enzyme conc. (50 g) Constant in a 500 l of reaction
mixture.
JPSI 2 (1), Jan Feb 2013, 53-56

Kumar A. et al: Pepsin Assay for Prescreening of HIV-Protease Inhibitors

soluble peptide of digested Hemoglobin. In our experiment
we have taken three crud plant extract to check the inhibitory
effect their extract in this assay system, and we observed
slight inhibitory effect with crud extract of zingiber officinale
while the enzymatic activity of pepsin were not affected by
the other (Aspargus recemosus, Aloe barbadensis) extracts.
We have taken paptsetin A as a negative control, which
natural inhibitor of this enzyme show approximately 99%
activity inhibition of this enzyme. The results are
reproducible and I hope that this assay system will encourage
the prescreening programme of HIV-1 protease.
ACKNOWLEDGEMENT
The author is very grateful to Professor G.S. Randhawa,
Head Department of Biotechnology, IIT ROORKEE, INDIA,
as they provide technical assistance during my study, and
also to Mr. Manish Rana, for their kind support.
Fig.2: Enzyme (Pepsin) kinetics keeping enzyme conc. Constant.

Finally we performed enzyme inhibition assay, here we take

three extract of plant sample that is Asparagus sp., zingiber
sp. and alovera sp. and activity of enzyme were observed. We
have also taken papestatin A as a negative control.

absorbance at 280 nm

0.4

0.3

0.2

0.1

0.0

Ex1

Ex2

Ex3

Experimental sample

Fig.-3 Blue color bar (C) shows activity of enzyme in the absence of any
inhibitor, negligible bar (I) shows maximum inhibition in the presence of
natural pepsin inhibitor (Papestatin A). Ex1, Ex2 and Ex3 bar shows enzyme
activity in the presence of different plant extract i.e. Aspargus recemosus,
Zingiber officinele, Aloe barbadensis respectively, and middle blue bar
shows the comparison of enzyme activity in the presence of plant extract
with control simultaneously.

RESULT AND DISCUSSION

The purpose of this work was to develop an assay system for
prescreening of HIV-1 protease inhibitors form crud plant
extracts, as the previous research shows that pepsin had great
similarities with HIV- protease in structure and function as
well, so we can use it as a cheapest substitute of HIVprotease. Many research groups had been developed different
kind of assay system with pepsin. Here we set different
parameters according to our requirements. Basic mechanism
behind this assay30 is that when we allowed reaction by
mixing enzyme and substrate in reaction vial left them for
incubation for few minute then enzyme pepsin cleaves
substrate hemoglobin into small pieces after incubation we
added TCA (Tri Chloro Acetic acid) to stop the reaction
simultaneously the large protein particles also get precipitated
but the digested protein i.e. Smaller peptide or cleaved
product of enzyme is still soluble in the reaction mixture and
we can remove undigested precipitated part leaving digested
part in the reaction mixture by centrifugation at 14000 g. and
we take absorbance at 280 nm, which comes due the presence
of tryptophan and tyrosin amino acids incorporated into the

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How to cite this article:

Singh K.P., Kumar A., Prasad R. Pepsin assay one of the easiest approach for prescreening of HIV-protease inhibitors. J Pharm Sci Innov. 2013; 2(1): 5356.

JPSI 2 (1), Jan Feb 2013, 53-56